A unified viscoplastic model for high temperature low cycle fatigue of service-aged P91 steel

The finite element (FE) implementation of a hyperbolic sine unified cyclic viscoplasticity model is presented. The hyperbolic sine flow rule facilitates the identification of strain-rate independent material parameters for high temperature applications. This is important for the thermo-mechanical fatigue of power plants where a significant stress range is experienced during operational cycles and at stress concentration features, such as welds and branched connections. The material model is successfully applied to the characterisation of the high temperature low cycle fatigue behavior of a service-aged P91 material, including isotropic (cyclic) softening and nonlinear kinematic hardening effects, across a range of temperatures and strain-rates

List of Primers used for semi-quantitative reverse transcriptase polymerase chain reaction.

<p>List of Primers used for semi-quantitative reverse transcriptase polymerase chain reaction.</p

Jasmonate profiles of <i>CYP94</i>-mutants after wounding.

<p>Plants were grown under short day conditions (8 h light / 16 h dark) at 22°C. Rosette leaves of six-week-old plants were wounded three times across the mid vein. Damaged rosette leaves were harvested for phytohormone extraction at indicated time points according to hours post wounding (hpw). Extracts of pooled rosette leaves were analyzed by LC-MS/MS. Quantitative data of the wounding time course are given in nmol/g FW for A) jasmonic acid (JA), B) jasmonic acid-isoleucine (JA-Ile), C) 12-hydroxy-jasmonic acid-isoleucine (OH-JA-Ile), D) 12-carboxy-jasmonic acid-isoleucine (COOH-JA-Ile) and E) 12-hydroxy-jasmonic acid (OH-JA). Each data point represents the mean value ± SD of four biological replicates. Asterisks indicate significant differences between Col-0 and mutant according to student’s t-test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). For a detailed one-way analysis of variance (using the Tukey post-hoc test, <i>p</i><0.05) we refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159875#pone.0159875.s008" target="_blank">S2 Table</a>.</p

High resolution MS² analysis of N-acetyl-amino adipate.

<p>UHPLC-ESI QTOF-MS fragmentation analysis of a metabolite marker accumulating in flowers with impaired CYP94B1 functionality. MS² spectrum of N-acetyl-amino adipate (<i>m/z</i> 202.0726, retention time 1.72 min) is shown for the negative ionization mode with a collision energy of 10 eV. Loss of the N-bound acetyl group leads to the fragment of <i>m/z</i> 160.0614. Subsequent losses of water and the carboxy-group of the amino adipate result in the fragments of <i>m/z</i> 142.0511 and <i>m/z</i> 106.0614.</p

Expression profile of promoter:GUS-constructs for CYP94B1, CYP94B2, CYP94B3 and CYP94C1 in vegetative organs of <i>A</i>. <i>thaliana</i>.

<p>Transformed plants were grown on soil under long-day (16 h light / 8 h dark) conditions. Seedlings were grown on ½ MS plates. All plant lines were stained with 2 mM X-Gluc. Staining was performed with two independent plant lines per construct with comparable results. Staining was performed ≥3 times with each line with comparable results.</p

Metabolite fingerprinting analysis of flowers of Col-0 and <i>CYP94</i>-mutant lines.

<p>Col-0 and <i>cyp94b1</i>, <i>cyp94b2</i>, <i>cyp94b3</i>, <i>cyp94c1</i> single, double, triple and quadruple mutant plants were grown under long day conditions (16 h light/8 h dark) at 22°C. Flowers were harvested at stage 13–14, homogenized, extracted by two-phase-extraction and analyzed by UHPLC/ESI-TOF MS. A subset of 164 high quality metabolite features (FDR < 10⁻⁴) derived from the positive ionization mode of the polar and the non-polar extraction phase were obtained. <b>A</b>) For metabolite-based clustering by means of one-dimensional self-organizing map (1D-SOM) 7 clusters were selected. The width of a cluster is proportional to the number of features assigned to the cluster. The heat map colors represent average intensity values (see color map right-hand side). For analysis two independent experiments with at least two pools of flowers each were used. Reliable features of both experiments were used for 1D-SOM representation. <b>B</b>) Relative amount of selected metabolite markers. Identities of the markers were confirmed by MS² analysis. Data analysis and visualization were performed with MarVis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159875#pone.0159875.ref025" target="_blank">25</a>].</p

High resolution MS² analyses of 12-carboxy-JA and 12-carboxy-JA-Ile.

<p>UHPLC-ESI QTOF-MS fragmentation analyses of metabolite markers depleted in flowers with impaired CYP94C1 functionality. MS² spectra of <b>A)</b> 12-carboxy-JA (<i>m/z</i> 239.0948, RT 3.53 min) and <b>B)</b> 12-carboxy-JA-Ile (<i>m/z</i> 352.1814, RT 4.50 min) are shown for negative ionization mode with a collision energy of 10 eV. The loss of the ω-carboxy group leads to the fragments of <i>m/z</i> 195.1051 for 12-carboxy-JA and <i>m/z</i> 308.1918 for 12-carboxy-JA-Ile. The free α-carboxy group of 12-carboxy-JA results in the fragment of <i>m/z</i> 59.0159, while 12-carboxy-JA-Ile shows the fragment of <i>m/z</i> 130.0899 for isoleucine.</p

List of Primers used for cloning of promoter-β-glucoronidase fusion constructs.

<p>List of Primers used for cloning of promoter-β-glucoronidase fusion constructs.</p

Pathway of JA activation and inactivation.

<p>Plant wounding induces the conversion of 18:3(n-3) to JA (for details see text). JA might be conjugated to Ile yielding JA-Ile, a reaction catalyzed by JAR1. JA-Ile is the bioactive phytohormone, which can be perceived by the SCF^COI-complex leading to the de-repression/induction of JA-responsive genes. Inactivation of JA-Ile signaling can be achieved via two possible routes: either by the enzymatic activity of the amido-hydrolases ILL6 and IAR3 that catalyze the hydrolytic cleavage of JA-Ile, or by enzymatic activity of distinct members of the cytochrome P450 subfamily CYP94 (<i>i</i>.<i>e</i>. CYP94B1, CYP94B3 and CYP94C1) that catalyze the sequential ω-oxidation of JA-Ile to 12-hydroxy-JA-Ile and 12-carboxy-JA-Ile. Although all three mentioned CYP94-enzymes have the capacity to catalyze the hydroxylation (mono-oxygenation) as well as the carboxylation (double oxygenation), they exhibit distinct catalytic specificities. Beside JA-Ile, oxidized JA-Ile derivatives may also serve as substrate for IAR3 (and Ill6) <i>in planta</i>. JAR, JASMONATE RESISTENT1; IAR3, IAA-ALA-RESISTENT3; ILL6, IAA-LEU RESISTENT-like6.</p

An Iron 13<i>S</i>-Lipoxygenase with an α-Linolenic Acid Specific Hydroperoxidase Activity from <i>Fusarium oxysporum</i>

<div><p>Jasmonates constitute a family of lipid-derived signaling molecules that are abundant in higher plants. The biosynthetic pathway leading to plant jasmonates is initiated by 13-lipoxygenase-catalyzed oxygenation of α-linolenic acid into its 13-hydroperoxide derivative. A number of plant pathogenic fungi (e.g. <i>Fusarium oxysporum</i>) are also capable of producing jasmonates, however, by a yet unknown biosynthetic pathway. In a search for lipoxygenase in <i>F. oxysporum</i>, a reverse genetic approach was used and one of two from the genome predicted lipoxygenases (FoxLOX) was cloned. The enzyme was heterologously expressed in <i>E. coli</i>, purified via affinity chromatography, and its reaction mechanism characterized. FoxLOX was found to be a non-heme iron lipoxygenase, which oxidizes C₁₈-polyunsaturated fatty acids to 13<i>S</i>-hydroperoxy derivatives by an antarafacial reaction mechanism where the bis-allylic hydrogen abstraction is the rate-limiting step. With α-linolenic acid as substrate FoxLOX was found to exhibit a multifunctional activity, because the hydroperoxy derivatives formed are further converted to dihydroxy-, keto-, and epoxy alcohol derivatives.</p></div